A CGA/EGFR/GATA2 positive feedback circuit confers chemoresistance in gastric cancer
Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao
Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao
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Research Article Gastroenterology Oncology

A CGA/EGFR/GATA2 positive feedback circuit confers chemoresistance in gastric cancer

Abstract

De novo and acquired resistance are major impediments to the efficacy of conventional and targeted cancer therapy. In unselected gastric cancer (GC) patients with advanced disease, trials combining chemotherapy and an anti-EGFR monoclonal antibody have been largely unsuccessful. In an effort to identify biomarkers of resistance so as to better select patients for such trials, we screened the secretome of chemotherapy-treated human GC cell lines. We found that levels of CGA, the α-subunit of glycoprotein hormones, were markedly increased in the conditioned media of chemoresistant GC cells, and CGA immunoreactivity was enhanced in GC tissues that progressed on chemotherapy. CGA levels in plasma increased in GC patients who received chemotherapy, and this increase was correlated with reduced responsiveness to chemotherapy and poor survival. Mechanistically, secreted CGA was found to bind to EGFR and activate EGFR signaling, thereby conferring a survival advantage to GC cells. N-glycosylation of CGA at Asn52 and Asn78 is required for its stability, secretion, and interaction with EGFR. GATA2 was found to activate CGA transcription, whose increase, in turn, induced the expression and phosphorylation of GATA2 in an EGFR-dependent manner, forming a positive feedback circuit that was initiated by GATA2 autoregulation upon sublethal exposure to chemotherapy. Based on this circuit, combination strategies involving anti-EGFR therapies or targeting CGA with microRNAs (miR-708-3p and miR-761) restored chemotherapy sensitivity. These findings identify a clinically actionable CGA/EGFR/GATA2 circuit and highlight CGA as a predictive biomarker and therapeutic target in chemoresistant GC.

Authors

Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao

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Figure 4

N-glycosylation is required for CGA-induced chemoresistance.

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N-glycosylation is required for CGA-induced chemoresistance. (A) Left: S...
(A) Left: SDS-PAGE of purified CGA from HEK293FT cells (rCGA) and E. coli (E. coli CGA). Right: Viability of CGA–/– SGC7901ADR cells treated with rCGA or E. coli CGA and chemotherapy. (B and C) Immunoblotting of lysate and conditioned medium from PNGase F–treated SGC7901ADR cells (B), with viability measured in indicated cells treated with chemotherapy (C). (D) MS/MS spectra of CGA secreted by SGC7901ADR cells shows 2 N-glycosylation sites, Asn52 (left) and Asn78 (right), in CGA. N in red indicates the glycosylation sites. (E) Viability of CGA–/– SGC7901ADR cells transfected with WT, N52Q, N78Q, or N52Q/N78Q double mutant (DM) CGA and treated with chemotherapy. (F) Immunoblotting of lysates and conditioned medium CGA from CGA–/– SGC7901ADR cells transfected with WT, N52Q, N78Q, or DM CGA. Asterisk and arrowhead indicate CGA band shifts. (G) Immunoblotting of lysate and conditioned medium CGA from MDR cells treated with BFA (5 nM). (H) Immunoblotting of CGA from CGA–/– SGC7901ADR cells transfected with WT, N52Q, N78Q, or DM CGA and treated with BMA (1 μM) or MG132 (10 μM). (I) Immunoblotting of p-EGFR and EGFR in CGA–/– SGC7901ADR cells treated with purified WT, N52Q, N78Q, or DM rCGA. (J) Immunoblotting of lysates from SGC7901 cells transfected with Flag-tagged EGFR were incubated with purified His-tagged CGA after immunoprecipitation with anti-Flag and anti-His antibodies. (K) Top: Immunoblotting for Flag of bound proteins after GST or GST fusion proteins were incubated with equal amounts of lysates from Flag-tagged EGFR-ECD–expressing HEK293T cells. Bottom: Ponceau-S staining to detect bait proteins. Arrowhead and asterisk indicate GST and GST fusion proteins, respectively. (L) IF staining of CGA, EGFR, and DAPI staining in SGC7901 cells treated with WT, N52Q, N78Q, or DM rCGA for 10 minutes at 4°C. Scale bar: 10 μm. Data are presented as mean ± SEM. **P < 0.01 by 1-way ANOVA with Dunnett’s multiple-comparison test (A, C, and E).